Substrate analogs that substitute for lipid I as a substrate for MurG

Abstract

General methods for monitoring the activity of MurG, a GlcNAc transferase involved in bacterial cell wall biosynthesis, is disclosed. More particularly, the synthesis of simplified substrate analogs of Lipid I (the natural substrate for MurG), which function as acceptors for UDP-GlcNAc in an enzymatic reaction catalyzed by MurG, is described. Assays using the substrate analogs of the invention are further disclosed, which are useful for identifying a variety of other substrates, including inhibitors of MurG activity, for facilitating mechanistic and / or structural studies of the enzyme and for other uses. High throughput assays are also described.

Description

1. FIELD OF THE INVENTIONThe present invention relates to substrate analogs of UDP-GlcNAc:muramyl pentapeptide pyrophosphoryl, N-acetylglucosaminyltransferase (GlCNAc transfer, MurG, or its homologs), an enzyme involved in a bacterial cell wall biosynthesis. The substrate analogs of the invention are useful as functional substitutes of Lipid I, the membrane bound, natural substrate of MurG. In particular, the substrate analogs of the present invention can be used advantageously in an assay for the enzymatic activity catalyzed by MurG, in methods for identifying other substrate analogs of MurG, as well as inhibitors of enzymatic activity or cell wall biosynthesis (i.e., potential antibacterial drugs), and in the isolation / purification of MurG, including studies of its active protein / peptide fragments.2. BACKGROUND OF THE INVENTION2.1 Bacterial EnzymologyThe emergence of resistance to existing antibiotics has rejuvenated interest in bacterial enzymology. It is hoped that detailed mech...

AI Technical Summary

Benefits of technology

Substrate analogs for MurG enzyme, a GlcNAc transferase, are disclosed. For the first time, a substrate analog of Lipid I, as shown above in Scheme I, (i) having a structure that is accepted by at least wild type MurG enzyme such that a labeled coupling product is produced by the GlcNAc transferase activity of the enzyme in the presence of the substrate analog and labeled UDP-GlcNAc, and (ii) having structural features that facilitate the separation of labeled UDP-GlcNAc from the labeled coupling product.

Problems solved by technology

While some detailed structural and mechanistic information on some of the early enzymes in the pathway is now available, most of the downstream enzymes have proven very difficult to study.

There are two main reasons for this difficult: First, the downstream enzymes are membrane-associated, making them intrinsically hard to handle; secondly, discrete substrates for most of the downstream enzymes are either not available or not readily so.

In some cases monomeric substrates are difficult to obtain in large quantities from natural sources.

In the absence of readily available discrete substrates, it has been impossible to develop enzyme assays that can be used to measure the activity of the downstream enzymes reliably and under a well-defined set of reaction conditions.

This unfulfilled need has thwarted attempts to purify many of the downstream enzymes in an active form suitable for structural characterization, much less permitted attempts to obtain detailed mechanistic information on such enzymes.

), the downstream enzymes have proven exceedingly difficult to study.

), making them intrinsically hard to handle, and partly because substrates for many of the enzymes are not readily available.

These problems have impeded the development of activity assays suitable for detailed mechanistic investigations of the downstream enzymes.

There are no mammalian homologs, and no direct assays for MurG activity have been developed, in part because the lipid-linked substrate (Lipid I, Scheme 1) is extremely difficult to isolate.

Although it is possible to increase the quantities of lipid-linked substrate by using cells engineered to overexpress enzymes involved in the synthesis of the lipid-linked substrate, isolation remains very difficult.

Moreover, the isolated substrate is hard to handle.

Although this "coupled" enzyme assay is manageable for screening of potential inhibitors of the MurG enzyme, it is not suitable for detailed mechanistic investigations, and it cannot be used to follow MurG activity during purification.

Despite decades of effort spent characterizing MurG activity, there is virtually no structural or mechanistic information on the enzyme.

Consequently, it has not been possible to purify MurG in a quantifiably active form or to determine the minimal functional length; not has it been possible to carry out any detailed mechanistic studies, or to determine the substrate requirements.

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